The present invention relates generally to the field of nucleotides. In particular, the invention relates to measuring the potential biological activity of a compound by measuring nucleotide levels in cells following chemical treatment, and more particularly it pertains to measuring the levels of cyclic phosphate nucleotides present in cells following chemical treatment for measuring the potential biological activity of a compound.
The physiological responses to many biologically active compounds are mediated through xe2x80x9csecond messengersxe2x80x9d. Nucleotides, for example cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate or cyclic AMP (cAMP), play important roles as second messengers in signal transduction pathways after hormones or other biologically active compounds bind to cell surface receptors. Increased levels of nucleotides, resulting from receptor activation, cause activation of specific nucleotide-dependent protein kinases, which in turn cause phosphorylation of various target proteins. It is the activation of these phosphorylated target proteins that bring about the diverse physiological responses in the cells including, but not limited to, biological activity. Thus, the higher the level of the nucleotide present the more biological activity a compound may possess.
The measuring of intracellular nucleotide levels following chemical treatment of cells has been reported in the literature, for example Takeda et al., J. Biochem., Vol. 105, pp. 327-329 (1989), Berg et al., Biotechniques, Vol. 15, No. 1, pp. 56-59 (1993), Brown et al., Biochem. J. 121, pp. 561-562 (1971), and A. G. Gilman, PNAS, Vol. 67, No. 1, pp. 305-312 (1970). However, these methods tend to involve multiple reaction vessels and be time-intensive limiting the number of measurements that can be carried out in a given time period. As such, these methods preclude a single vessel, i.e. xe2x80x9cone-potxe2x80x9d, high-throughput method for measuring changes in nucleotides. In addition, xe2x80x9cone-potxe2x80x9d methods reported in the literature, for example Amersham LifeScience""s commercially available Biotrak(trademark) product and Kariv et al., J. Biomolecular Screening, Vol. 4, No. 1, pp. 27-32, (1999), tend to be expensive. As a result, there is a need for an inexpensive, single vessel, high-throughput method for measuring changes in the amount of a nucleotide present in a cell.
One embodiment of the present invention describes a single vessel, high-throughput method for measuring levels of nucleotides generated in a testing medium. The present invention measures changes in the amount of a nucleotide in a test medium in response to the addition of a test compound to the test medium. The present invention is particularly effective in measuring changes in a nucleotide, such as cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate (cAMP), in a cell, particularly in cells of insects.
In another embodiment of the present invention, a single vessel, high-throughput method of identifying compounds that which increase the amount of a nucleotide generated by a testing medium by comparing test compounds to the test medium alone or to the test medium following chemical treatment with compounds that increase nucleotides is disclosed. This method can be useful in identify compounds suspected of exhibiting biological activity.
In yet another embodiment of the present invention, a single vessel, high-throughput method of identifying compounds with biological activity through the generation of cAMP in a cell is disclosed.
The present invention is less complex, more cost effective, and comparable in sensitivity to those disclosed in the art.
The modifier xe2x80x9caboutxe2x80x9d as utilized herein indicates that certain preferred operating ranges, such as ranges for molar ratios for reactants, material amounts, and temperature, are not fixedly determined. The meaning will often be apparent to one of ordinary skill in the art of molecular biology. For example, a recitation of a temperature range of about 120xc2x0 C. to about 135xc2x0 C. in reference to an experiment would be interpreted to include other like temperatures that can be expected to favor a useful completion of the experiment, such as 105xc2x0 C. or 150xc2x0 C. In general, unless more particular ranges are disclosed, xe2x80x9caboutxe2x80x9d shall indicate not more than 10% of the absolute value of an end point or 10% of the range recited, whichever is less.
The term xe2x80x9cambient temperaturexe2x80x9d as utilized herein shall mean any suitable temperature found in a laboratory or other working quarter, and is generally not below about 15xc2x0 C. nor above about 30xc2x0 C.
The term xe2x80x9cbiological activityxe2x80x9d as utilized herein shall mean the ability of a substance, such as a chemical, including but not limited to drugs (i.e. pharmaceuticals) and pesticides, to act on a cell, virus, organ or organism and which creates a change in the functioning of the cell, virus, organ or organism.
The term xe2x80x9ctesting vesselxe2x80x9d as utilized herein shall mean any device, such as a petri-dish, a microtiter plate, a test-tube, or beaker, which may be utilized to perform an assay, a reaction, a method, an experiment, or other procedure.
The term xe2x80x9ctesting mediumxe2x80x9d as used herein shall mean any environment, such as a cell or a cellular membrane, suitable for generating a nucleotide.
The term xe2x80x9cnucleotide binding proteinxe2x80x9d as utilized herein shall mean any protein, for example, a protein derived from a bovine adrenal gland, a protein derived from a bovine muscle, or an antibody, that selectively binds or attaches to a nucleotide.
As used herein, the term xe2x80x9clysing agentxe2x80x9d shall mean any substance, such as a detergent, capable of causing cell lysis.
One embodiment of the present invention involves a method of measuring levels of a nucleotide generated in a testing medium, for example, cells or cellular membranes, following chemical treatment. The method comprises:
(a) contacting a test compound with the testing medium in a testing vessel, for example, a microtiter plate, a test-tube, or beaker;
(b) maintaining the test compound in contact with the testing medium in the testing vessel for a time sufficient to allow nucleotides to be generated in the testing medium;
(c) releasing nucleotides: generated in the testing medium into the testing vessel;
(d) adding a radiolabeled nucleotide ligand and a fixed amount of a nucleotide binding protein to the testing vessel, wherein the radiolabeled nucleotide ligand competes with nucleotides generated in the testing medium to bind to the nucleotide binding protein;
(e) maintaining the testing vessel for a period of time at a temperature sufficient to allow nucleotides generated in the testing medium and the radiolabeled nucleotide ligand to bind to the nucleotide binding protein to form a nucleotide binding protein complex;
(f) separating the nucleotide binding protein complex from uncomplexed radiolabeled nucleotide ligand; and
(g) measuring the level of radioactivity of the nucleotide binding protein complex, wherein the level of radioactivity is inversely proportional to the amount of the nucleotide generated in said testing medium.
Suitable nucleotides that may be generated and measured by the present invention include, but are not limited to, cyclic phosphates. Preferable nucleotides generated and measured by the present invention are cyclic monophosphates, for example, cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate (cAMP), dibutyryl cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate (dbcAMP), cyclic guanosine 3xe2x80x2,5xe2x80x2-monophosphate (cGMP), cyclic inosine 3xe2x80x2,5xe2x80x2-monophosphate (cIMP), and cyclic uridine 3xe2x80x2,5xe2x80x2-monophosphate(cUMP). Particularly preferred and useful nucleotides generated and measured by the present invention are cAMP and cGMP.
Suitable cells that may be used as the test medium in the present invention include, but are not limited to, native or cloned invertebrate or vertebrate cells which are either adherent or nonadherent, for example Sf9 cells, Sf21 cells, KC cells, CHO cells, COS7 cells, and HEK293 cells.
The test compound may be contacted neat or as a solution in a solvent. As used herein the term xe2x80x9cneatxe2x80x9d refers to the unmixed or straight technical material along with any impurities contained therein. Examples of solvents which may be used in the present invention are saline solutions, for example potassium, sodium, or magnesium saline solutions, a tissue culture media, buffers, for example acidic, basic, or neutral buffers, water, an acid, a ketone, an alcohol, a sulfoxide, or mixtures thereof. Preferably test compound is added as a solution in a tissue culture media, N,N-dimethylsulfoxide, methanol, acetone, or mixtures thereof. The test medium can also be added neat or as a solution in a solvent. The solvents set forth above may also be used in connection with the test medium.
The time sufficient to allow nucleotides to be generated in the testing medium is preferably from about five to 180, more preferably about five to about sixty, minutes, at about 15xc2x0 C. to about 40xc2x0 C., more preferably about 20xc2x0 C. to about 40xc2x0 C.
The nucleotides generated in the testing medium may be released into the testing vessel by methods known to one skilled in the art. Preferably, the nucleotide can be released into the testing vessel by lysing the test medium, for example, through the addition of a lysing agent, either neat or as a solution in the above disclosed solvents; mechanically disrupting the cell wall, for example, using ultrasound; freeze-thawing; heating; or acid and alkaline treatment; and then maintaining the testing medium at about 15xc2x0 C. to about 40xc2x0 C., preferably about 20xc2x0 C. to about 40xc2x0 C., for about 5 to about sixty, preferably for about 5 to about thirty, minutes. Preferably the testing medium is lysed through the addition of a lysing agent, such as a nonionic detergent, enzyme, or surfactant.
Preferably, the radiolabeled nucleotide ligand is a radiolabeled cyclic phosphate, more preferably, a radiolabeled cyclic monophosphate. Examples of radiolabeled nucleotide ligands that may be used include, but are not limited to, radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate, radiolabeled cyclic guanosine 3xe2x80x2,5xe2x80x2-monophosphate, radiolabeled cyclic inosine 3xe2x80x2,5xe2x80x2-monophosphate, or radiolabeled cyclic uridine 3xe2x80x2,5xe2x80x2-monophosphate. Preferred radiolabeled nucleotide ligands that may be used in the present invention are radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate and radiolabeled cyclic guanosine 3xe2x80x2,5xe2x80x2-monophosphate, more preferably radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate.
As set forth above, examples of nucleotide binding proteins that may be useful in the present invention are proteins derived from a bovine adrenal gland and muscle and antibodies. A preferred nucleotide binding protein that can be used in the present invention is a protein derived from a bovine adrenal gland. The time required for the nucleotides generated in the testing medium and the radiolabeled nucleotide ligand to bind to the nucleotide binding protein to form a nucleotide binding protein complex is not critical to the present invention. In general, maintaining the testing vessel at about 15xc2x0 C. to about 40xc2x0 C., preferably about 20xc2x0 C. to about 40xc2x0 C., for about 15 to about 120, preferably about 90 to about 120, minutes, is sufficient to allow nucleotides generated in the testing medium and the radiolabeled nucleotide ligand to bind to the nucleotide binding protein to form a nucleotide binding protein complex.
The nucleotide binding protein complex can be separated from the uncomplexed radiolabeled nucleotide ligand by methods known to one skilled in the art, such as filtration, centrifugation, solvent extraction or combinations thereof. Similarly, the level of radioactivity of the nucleotide binding protein complex, which is inversely proportional to the amount of the nucleotide generated, can be measured by methods known to one skilled in the art, such as scintillation counting or spectrophotometric methods (see, for example, A. G. Gilman, PNAS, Vol. 67, No. 1, pp. 305-312 (1970) incorporated herein by reference to the extent it discusses measuring the radioactivity of a complex the inverse relationship between the level of radioactivity and the amount of the nucleotide generated in a cell).
In another embodiment of the present invention, a method of identifying a compound that increases the amount of a nucleotide generated by a testing medium is disclosed. The method comprises performing a trial utilizing the method disclosed above and then comparing the results form the trial to results produced from either:
(a) a negative control in which no compound is contacted with the testing medium;
(b) appositive control using a positive control compound as the test compound, wherein the positive control compound is a compound that increases the amount of the nucleotide generated in a cell; or
(c) both a positive control and a negative control; wherein an amount of nucleotide generated in the testing medium is greater than the nucleotide that appears in the testing medium in the negative control and an amount of nucleotide generated in the testing medium is greater than or equal to the amount of nucleotide generated in the testing medium in the positive control is indicative of a test compound which can increase the amount of nucleotide generated in a testing medium.
Preferably, the results from the trial are compared to results produced from both a positive control and a negative control; wherein an amount of nucleotide generated in the testing medium is greater than the nucleotide that appears in the testing medium and an amount of nucleotide generated in the testing medium is greater than or equal to the amount of nucleotide generated in the testing medium in the positive control is indicative of a test compound which can increase the amount of nucleotide generated in a testing medium.
Examples of positive control compounds that can be used in the present invention are octopamine, synephrine, demethylchlordimeform, or amitraz.
The method is particularly useful identifying compounds that exhibit biological activity. The testing mediums, nucleotides, testing vessels, radiolabeled nucleotide ligands, and nucleotide binding proteins including, but not limited to, the preferred testing mediums, nucleotides, testing vessels, radiolabeled nucleotide ligands; and nucleotide binding proteins disclosed above can also be used in this embodiment.
As set forth above, the time sufficient to allow nucleotides to be generated in the testing medium is about five to 180, preferably about five to sixty, minutes at about 15xc2x0 C. to about 40xc2x0 C., preferably about 20xc2x0 C. to about 40xc2x0 C.
Similarly, the nucleotides generated in the generated in the testing medium can be released into the testing vessel by the methods disclosed above, which are incorporated herein as if set forth at length.
As set forth above, maintaining the testing vessel at about 15xc2x0 C. to about 40xc2x0 C., preferably about 20xc2x0 C. to about 40xc2x0 C., for about 15 to about 120,preferably about 90 to about 120, minutes, is usually sufficient to allow nucleotides generated in the testing medium and the radiolabeled nucleotide ligand to bind to the nucleotide binding protein to form a nucleotide binding protein complex. The methods of separating the binding protein complex from uncomplexed radiolabeled nucleotide ligand and measuring level of radioactivity of the nucleotide binding protein complex set forth above can also be used in this embodiment.
In another embodiment of the present invention, a method of identifying a compound with biological activity is disclosed. The method comprises:
i) performing a trail comprising the steps of:
(a) contacting a test compound with a cell in a microtiter plate;
(b) maintaining the test compound in contact with the cell in the microtiter plate for a time sufficient to allow cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate to be generated in the cell;
(c) releasing cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in the cell into the microtiter plate;
(d) adding radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-mohophosphate and a fixed amount of a protein derived from a bovine adrenal gland to the microtiter plate, wherein the radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate competes with cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in the cell to bind to the protein derived from a bovine adrenal gland;
(e) maintaining the microtiter plate at a temperature for a period of time sufficient to allow cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in the testing medium and the radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate to bind to the protein derived from a bovine adrenal gland to form a cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate binding protein complex;
(f) separating the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate binding protein complex from uncomplexed radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate; and
(g) measuring the level of radioactivity of the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate, binding protein complex, wherein the level of radioactivity is inversely proportional to the amount of the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in said cell; and
ii) comparing the results from the trial to results produced from either:
(a) a negative control in which no compound is contacted with the cell;
(b) a positive control using a positive control compound as the test compound, wherein the positive control compound is a compound that increases the amount of the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in a cell; or
(c) both a positive and a negative control; wherein an amount of cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in the testing medium is greater than the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate that appears in the cell in the negative control and an amount of cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in the cell is greater than o equal to the amount of cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in the cell in the positive control is indicative of a test compound which can increase the amount of cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in a cell.
The positive control compounds disclosed above can also be used in this embodiment. Preferably, the positive control compound is octopamine or amitraz.
Preferably, the time sufficient to allow cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate to be generated in the cell is about five to 180, more preferably about five to about sixty, minutes at about 15xc2x0 C. to about 40xc2x0 C., more preferably about 20xc2x0 C. to about 40xc2x0 C. Preferably, the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in the cell is released into the microtiter plate by lysing, preferably through the addition of a lysing agent, such as a nonionic detergent, and maintaining the cell at about 15xc2x0 C. to about 40xc2x0 C., more preferably about 20xc2x0 C. to about 40xc2x0 C., for about 5 to about sixty, more preferably about 5 to about thirty, minutes.
Generally, the microtiter plate can be maintained for about 15 to about 120, preferably about 90 to about 120, minutes at about 15xc2x0 C. to about 40xc2x0 C., preferably about 20xc2x0 C. to about 40xc2x0 C., to allow cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate generated in the cell and the radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate to bind to the protein derived from a bovine adrenal gland to form the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate binding protein complex. Once the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate binding protein complex is formed it can be separated form the uncomplexed radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate and its radioactivity measured by the methods disclosed above. Preferably, the cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate binding protein complex is separated from the uncomplexed radiolabeled cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate by filtration and its radioactivity is measured by scintillation counting.
The present invention provides an improvement over other methods disclosed in the art in that it is a single vessel, high-throughput method for measuring increases in the amount of a nucleotide generated as well as a means of identifying compounds with biological activity which is less complex, more cost effective, yet comparable in sensitivity to those disclosed in the art.